Synthesis, Characterization and In-vitro Evaluation of Novel Naphthoquinone Derivatives and Related Imines: Identification of New Anticancer Leads.

Quinones such as 1,4-naphthoquinones are abundant in nature and naphthoquinone based natural products are known to possess anticancer activity. This pharmacophore is known to convey anticancer activity to some drugs such as streptonigrin, mitomycin A, etc. We synthesized and characterized different classes of naphthoquinone derivatives including bis naphthoquinone, 2-arylaminonaphthoquinone, benzoxantene-6,11-dione and benzoacridine-5,6-dione derivatives instead of the expected 2-hydroxy-3-(substituted phenyl(aryl amino)methyl)naphthalene-1,4-dione derivatives from the reaction of 2-hydroxy1,4-naphthoquinone (lawson) with different benzaldehydes and aryl amines. Benzoacridine-5,6-dione derivatives and related imines showed potent anti-breast cancer activity in MCF-7 cancer cells. The in-vitro results revealed that five compounds benzoacridinedione derivatives (6b and 7b) and imines (13, 14 and 15) by the IC50 range of 5.4-47.99 μM are the most potent anti-breast cancer structures.


Introduction
Quinones have been frequently exploited for the discovery of cellular mechanisms associated with cytotoxicity in various cancer cells. The redox properties of quinones can often induce apoptosis in cancer cells through oxidative stress induced by the in-situ creation of reactive oxygen species (ROS), while additional evidence proposes that some quinones can intercalate with DNA or inhibit proteins involved in DNA replication (1). Quinones such as 1,4-naphthoquinones are abundant in nature (1) and naphthoquinone based natural products are known to possess anticancer activity. This pharmacophore is known to convey anticancer activity to some drugs such as streptonigrin, mitomycin A, etc (2, 3). 1,4-Naphthoquinones are extensively distributed in nature and many well-known important anticancer drugs having a quinone moiety such as anthracyclines, mitoxantrones, and saintopin ( Figure 1) have shown tremendous anticancer activity (4).
The naturally occurring naphthoquinone lapachol is the most plentiful naphthoquinoidal compound isolated from the core of the trees of the family Bignoniaceae. This natural product has been widely studied due to its important biological activities, including antitumoral (5, 6). β-lapachone is the most favorable molecule of the lapachol group. It is cytotoxic to a variety of human cancer cells (7), which are naturally more prone to oxidative damage in comparison to normal cells (8). β-lapachone has been widely studied in recent years and is now in phase II clinical trials as a monotherapy or in combination with other antitumor drugs (9).
On the other hand, the discovery of heat shock protein 90 (Hsp90) as the target of anticancer activity of geldanamycin ( Figure 2) has been attracted much attention in inhibition of Hsp90 as a tactic for the treatment of cancer. This led to huge efforts to develop clinically practical small Hsp90 inhibitor molecules (10-12) with a wide structural diversity including purinebased analogues (PU3) (11), naphthoquinone based structures (13,14), and resorcinol based structures (radicicol is a natural resorcinol derivative, Figure 2) (15,16).
In this study, we designed some 2-hydroxy-3-(substituted phenyl(aryl amino)methyl) naphthalene-1,4-dione derivatives possessing naphthoquinone and resorcinol rings as potential Hsp90 inhibitors. Furthermore, we designed related imines, which are lacking in naphthoquinone moiety, to investigate the role of naphthoquinone ring in biological activity of the designed naphthoquinone derivatives ( Figure 2). We characterized the structures of the prepared products by 1 H-NMR 13 ,C-NMR, Mass spectrometry, and CHN analysis. Surprisingly, what we separated was different from the expected 2-hydroxy-3-(substituted phenyl (aryl amino) methyl) naphthalene-1, 4-dione derivatives. The synthesized compounds were evaluated for their cytotoxic activity towards MCF-7 and PC3 cancer cell lines to find new anticancer leads.

General
All chemicals, reagents, and solvents were purchased from Merck AG and Aldrich Chemical. Melting points were determined on a Thomas-Hoover capillary apparatus. Infrared spectra were taken on a Perkin Elmer Model 1420 spectrophotometer in KBr pellets. 1 H and 13 C-NMR spectra were recorded on a Bruker FT-300 and 400 (Bruker Biosciences, USA) instrument at 300 (and 400) MHz and 75 MHz respectively, in DMSO-d 6 or CDCl 3 as solvents. The chemical shifts are recorded in compound isolated from the core of the trees of the family Bignoniaceae. This natural product has been widely studied due to its important biological activities, including antitumoral (5, 6). βlapachone is the most favorable molecule of the lapachol group. It is cytotoxic to a variety of human cancer cells (7), which are naturally more prone to oxidative damage in comparison to normal cells (8). β-lapachone has been widely studied in recent years and is now in phase II clinical trials as a monotherapy or in combination with other antitumor drugs (9). On the other hand, the discovery of heat shock protein 90 (Hsp90) as the target of anticancer activity of geldanamycin ( Figure 2) has been attracted much attention in inhibition of Hsp90 as a tactic for the treatment of cancer. This led to huge efforts to develop clinically practical small Hsp90 inhibitor molecules (10-12) with a wide structural diversity including purine-based analogues (PU3) (11), naphthoquinone based structures (13,14), and resorcinol based structures (radicicol is a natural resorcinol derivative, Figure 2) (15,16).
In this study, we designed some 2-hydroxy-3-(substituted phenyl(aryl amino)methyl) naphthalene-1,4-dione derivatives possessing naphthoquinone and resorcinol rings as potential Hsp90 inhibitors. Furthermore, we designed related imines, which are lacking in naphthoquinone moiety, to investigate the role of naphthoquinone ring in biological activity of the designed Synthesis of Novel Naphthoquinones as Anticancer Agents ppm relative to tetramethylsilane as internal standard. Coupling constant (J) values are in hertz (Hz) and spin multiples are given as s (singlet), d (double), t (triplet), q (quartet), and m (multiplet). Mass spectral data were recorded on a 6410 Agilent LCMS triple quadrupole mass spectrometer (LCMS) with an electrospray ionization (ESI) interface.

General
All chemicals, reagents, and solvents were purchased from Merck AG and Aldrich Chemical.
Melting points were determined on a Thomas-Hoover capillary apparatus. Infrared spectra were taken on a Perkin Elmer Model 1420 spectrophotometer in KBr pellets. 1 H and 13 C-NMR spectra were recorded on a Bruker FT-300 and 400 (Bruker Biosciences, USA) instrument at 300 (and 400) MHz and 75 MHz respectively, in DMSO-d6 or CDCl3 as solvents. The chemical shifts are

Chemistry
One of the required substrate, 5-chloro-2,4dihydroxy benzaldehyde (1c), was synthesized following a similar procedure described by Gupta et al. (21). Chlorination of the aromatic ring of 1a was carried out using sodium hypochlorite solution in the basic aqueous medium (Scheme 1). But, (the observation of two doublet signals related to the two vicinal aromatic hydrogens instead of two singlet signals in 1 H-NMR spectrum, supports that the achieved aldehyde is 3-chloro-2,4-dihydroxybenzaldehyde (1b)). The revealed 1 H-NMR data as well as those pertaining to the reported structures possessing this aldehyde moiety in this work, conformed 3-chloro-2,4dihydroxybenzaldehyde (1b) instead; emerging two doublet signals related to the two vicinal aromatic hydrogens instead of two singlet dihydroxybenzaldehyde (1b) instead; emerging two doublet signals related to the two vicinal aromatic hydrogens instead of two singlet signals. We used 2,4-dihydroxybenzaldehyde (1a), 3chloro-2,4-dihydroxybenzaldehyde (1b), and 4-hydroxybenzaldehyde (1d) as the starting materials in the next reaction.
As illustrated in Scheme 2, in order to achieve the synthesis of the desired compounds, Mannich reaction was employed in the presence of indium chloride as a catalyst in ethanol under reflux according to the reported procedure (22). Scheme 2. The starting materials (1-3) and some of the expected products (4).
As illustrated in Scheme 2, in order to achieve the synthesis of the desired compounds, Mannich reaction was employed in the presence of indium chloride as a catalyst in ethanol under reflux according to the reported procedure (22).
As shown in Scheme 2, the expected product from the reaction using 1d, 2, and 3b as the starting materials was 4b and in the case of applying 1d, 2, and amine 3c, the formation of 4c was expected. However, one aromatic hydrogen loss in the 1 H-NMR data did not accommodate with these structures. This evidence suggests the formation of compounds 6a and 7a (entries 2 and 3 in Table 1) in which a new ring is established. On the other hand, the pattern of signals related to the aromatic hydrogens of naphthoquinone in 1 H-NMR spectra demonstrated a noticeable change compared to the other structures containing naphthoquinone: further deshielding of one of the aromatic hydrogen in comparison with the other three ones suggests the structures 6b and 7b. As a matter of fact, the presence of two carbonyl groups in an ortho arrangement in the vicinity of one H atom of naphthoquinone ring, leads to shift its signal to the downfield to a greater degree. Moreover, the very two close carbonyl resonances at 180.22 and 180.26 ppm, which are the very adjacent chemical shifts for the carbonyl 13 C-NMR signals, propose an orthoquinone moiety which is seen in the structure of 6b. A similar mechanism to that of Zhang et al. reported (24), describing the formation of benzoacridine-5,6-dione derivatives, is given in Scheme 4. Recently, the synthesis of different benzo[h]quinoline-5,6-dione derivatives and related compounds using 2-hydroxynaphthalene-1,4-dione have been reported (2, 24-28). It is noteworthy that the reaction in entry 2 in Scheme 1. Synthesis of 3-chloro-2,4-dihydroxybenzaldehyde (1b).
As illustrated in Scheme 2, in order to achieve the synthesis of the desired compounds, Mannich reaction was employed in the presence of indium chloride as a catalyst in ethanol under reflux according to the reported procedure (22).
In order to synthesize 4a (Scheme 2), a reaction of 4-hydroxybenzaldehyde (1d), 2hydroxynaphthalene-1,4-dione (2), and 4H-1,2,4-triazol-4-amine (3a) was carried out in the presence of InCl3 as a catalyst in ethanol under reflux. The obtained product was a bis-Scheme 2. The starting materials (1-3) and some of the expected products (4).  6-dione, 6b). The 1 H-NMR signals proposed compound 6b as the only product of this reaction, and the angular analogue 6c was not obtained. A possible reason for the formation of 6b can be attributed to the less steric hindrance (29). On the whole, it can be concluded that the formation of bis naphthoquinone and benzoacridine-5,6dione derivatives is as a result of competition between naphthoquinone and aryl amine in a nucleophilic attack to the intermediate X (3-(4-hydroxybenzylidene)naphthalene-1,2,4(3H)trione), see entries 1-3 in Table 1).
Another two classes of naphthoquinone derivatives achieved in the case of using ortho hydroxyl-containing aldehydes 1a and 1b, are 2-arylaminonaphthoquinone and benzoxanthene-6,11-dione derivatives (see entries 4-9 in Scheme 3. Proposed mechanisms for the formation of bisnaphtoquinone 5. As shown in Scheme 2, the expected product from the reaction using 1d, 2, and 3b as the starting materials was 4b and in the case of applying 1d, 2, and amine 3c, the formation of 4c was expected. However, one aromatic hydrogen loss in the 1 H-NMR data did not accommodate with these structures. This evidence suggests the formation of compounds 6a and 7a (entries 2 and 3 in Table 1)  Moreover, the very two close carbonyl resonances at 180.22 and 180.26 ppm, which are the very adjacent chemical shifts for the carbonyl 13 C-NMR signals, propose an ortho-quinone moiety which is seen in the structure of 6b. A similar mechanism to that of Zhang et al. reported (24), describing the formation of benzoacridine-5,6-dione derivatives, is given in Scheme 4. Recently, the synthesis of different benzo[h]quinoline-5,6-dione derivatives and related compounds using 2-hydroxynaphthalene-1,4-dione have been reported (2, 24-28). It is noteworthy that the reaction in entry 2 in Table 1 could be accomplished in two probable cyclization pathways leading to give  Table 1).

Scheme 4.
Proposed mechanism for the formation of 6b and 7b.
Another two classes of naphthoquinone derivatives achieved in the case of using ortho hydroxylcontaining aldehydes 1a and 1b, are 2-arylaminonaphthoquinone and benzoxanthene-6,11-dione derivatives (see entries 4-9 in Table 1). As amine 3b participated in the reaction, the product is merely the derivative of 2-arylaminonaphthoquinone 8 (see entry 4 in Table 1). In the case of applying amine 3d and 3c, the other novel product, benzoxanthene dione derivative (10 and 12) was obtained, as well. A reasonable possibility for the formation of 2-arylaminonaphthoquinones Scheme 4. Proposed mechanism for the formation of 6b and 7b. Table 1). As amine 3b participated in the reaction, the product is merely the derivative of 2-arylaminonaphthoquinone 8 (see entry 4 in Table 1). In the case of applying amine 3d and 3c, the other novel product, benzoxanthene dione derivative (10 and 12) was obtained, as well. A reasonable possibility for the formation of 2-arylaminonaphthoquinones (8, 9 and 11) is shown in Scheme 5. The probable initial formation of hydrogen bonding between hydroxyl group of naphthoquinone and amino group makes the C2 of lawson more electrophilic which facilitates the reaction through Michael addition mechanism (30). The probable sequence of steps leading to formation of benzoxanthenedione derivatives is given in Scheme 6.
First, the hydroxyl group of naphthoquinone is deprotonated by the amine favoring the nucleophilic attack of naphthoquinone into the aldehyde and the formation of intermediate X. Afterwards, subsequent Michael addition of naphthoquinone to the intermediate X and eliminating one water molecule afforded the corresponding products 10 and 12. As a matter of fact, the formation of benzoxanthenedione products is due to the ortho hydroxyl group of aldehyde which leads to the conversion of bisnaphthoquinone (a symmetric system) into benzoxanthenedione (nonsymmetric system) derivatives. This symmetric and nonsymmetric system can obviously be seen in the 1 H-NMR patterns of the aromatic hydrogens of two lawson molecules in the products (compare 1 H-NMR data of 5 as a symmetric bisnaphthoquinone analogue with 10 as a nonsymmetric bisnaphthoquinone analogue). Furthermore, Pelageev et al. (31) reported the formation of Scheme 5. Proposed mechanism for the formation of 2-arylaminonaphthoquinones (8, 9 and 11).
The probable initial formation of hydrogen bonding between hydroxyl group of naphthoquinone and amino group makes the C2 of lawson more electrophilic which facilitates the reaction through Michael addition mechanism (30). The probable sequence of steps leading to formation of benzoxanthenedione derivatives is given in Scheme 6. Scheme 6. Proposed mechanism for the formation of benzoxanthene-6,11-dione derivatives (10 and 12).
First, the hydroxyl group of naphthoquinone is deprotonated by the amine favoring the nucleophilic attack of naphthoquinone into the aldehyde and the formation of intermediate X.
Afterwards, subsequent Michael addition of naphthoquinone to the intermediate X and eliminating one water molecule afforded the corresponding products 10 and 12. As a matter of fact, the formation of benzoxanthenedione products is due to the ortho hydroxyl group of Scheme 5. Proposed mechanism for the formation of 2-arylaminonaphthoquinones (8, 9 and 11).
The probable initial formation of hydrogen bonding between hydroxyl group of naphthoquinone and amino group makes the C2 of lawson more electrophilic which facilitates the reaction through Michael addition mechanism (30). The probable sequence of steps leading to formation of benzoxanthenedione derivatives is given in Scheme 6. Scheme 6. Proposed mechanism for the formation of benzoxanthene-6,11-dione derivatives (10 and 12).
First, the hydroxyl group of naphthoquinone is deprotonated by the amine favoring the nucleophilic attack of naphthoquinone into the aldehyde and the formation of intermediate X.
Afterwards, subsequent Michael addition of naphthoquinone to the intermediate X and eliminating one water molecule afforded the corresponding products 10 and 12. As a matter of fact, the formation of benzoxanthenedione products is due to the ortho hydroxyl group of Scheme 6. Proposed mechanism for the formation of benzoxanthene-6,11-dione derivatives (10 and 12). 7,10-dihydroxy-12H-benzo[b]xanthene-6,11dione derivatives resulted from the reaction of hydroxy naphthazarins and o-vanillin containing a hydroxyl group at position 2 under mild acid catalysis (acid catalyzed condition). On the other hand, the formation of benzoacridine-5,6-dione derivatives (compare the obtained products of entries 2 and 3 with those of entries 4-9 in Table 1) through the reaction of benzaldehydes possessing hydroxyl group in ortho position (1a and 1b) with 2-hydroxy 1,4-naphthoquinone 2 and aryl amines (3b and 3c) did not occur. This can be most probably due to the more electron donating character of 1a and 1b in comparison to 1d and can be attributed to the mechanism of formation of benzoacridine-5,6dione derivatives (see Scheme 4).

In-vitro cytotoxic effects
The MTT assay was employed for the assessment of the cytotoxicity of prepared compounds to find new lead compounds in terms of antitumor activity.
Various Schiff bases were reported as Hsp90 inhibitors and anticancer agents (16, 21, 31 and 34) and herein, three novel Schiff bases of 3-chloro-2, 4-dihydroxybenzaldehyde (13, 14 and 15) also showed high cytotoxicity in MCF-7 cells. All three imines are possessing resorcinol ring which is an essential pharmacophore for character of 1a and 1b in comparison to 1d and can be attributed to the mechanism of formation of benzoacridine-5,6-dione derivatives (see Scheme 4).

In-vitro cytotoxic effects
The MTT assay was employed for the assessment of the cytotoxicity of prepared compounds to find new lead compounds in terms of antitumor activity.
Bis naphthoquinone (5) and benzoxantene-6,11-dione (10 and 12) derivatives which are all possessing 1,4-naphthoquinone moiety, did not exhibit significant cytotoxicity. One possible reason could be attributed to their high polarity leading to poor permeability to the lipophilic cell membranes. Generally our compounds did not show significant cytotoxic effects on PC3 cells in the concentration below 100 µM. Overall, benzoacridine-5,6-dione derivatives and imines (6b, 7b, 13, 14 and 15) showed stronger cytotoxic effects on PC3 cells compared to the other compounds. Benzoacridine-5,6-dione derivatives (6b and 7b) proved to be promising cytotoxic agents and have encouraged further evaluation of this scaffold. Additional work is in progress in an attempt to find more potent antitumor agents.

Conclusion
In summary, we synthesized and characterized novel naphthoquinone derivatives which can be classified in four different classes including bis naphthoquinone, 2-arylaminonaphthoquinone, benzoxantene-6,11-dione and benzoacridine-5,6-dione derivatives. Lawson has been used as the starting material for the synthesis of a variety of biologically active compounds and materials with significant properties. In organic synthesis, it has been used in many reactions (17) and to the best of our knowledge, it is the first report of characterization of four different scaffolds from the reaction of lawson with different arylamines and benzaldehydes. On the whole, it can be concluded that the formation of bis naphthoquinone and benzoacridine-5,6-dione derivatives is as a result of competition between naphthoquinone and aryl amine in a nucleophilic Compound concentration required to inhibit tumor cell proliferation by 50%. Data are presented as the mean ± SEM from the dose− response curves of three independent experiments. b The mean cell viability (%) ± SEM from the dose−response curves of three independent experiments at 100 μM concentration.
attack to the intermediate X (see the products of entries 1-3 in Table 1). The formation of benzoxanthenedione products is due to the ortho hydroxyl group of aldehyde which leads to the conversion of bisnaphthoquinone (a symmetric system) into benzoxanthene dione (nonsymmetric system) derivatives. We have studied the in-vitro anti-cancer activity of these compounds against MCF-7 and PC3 cell lines by MTT test. The in-vitro results revealed that five compounds benzoacridinedione derivatives (6b and 7b) and imine (13, 14 and 15) by the IC 50 range of 5.4-47.99 μM are the most potent antibreast cancer structures. (8) (10)